ABSTRACT
Objective To construct EHEC△z5150 using Red recombination and explore its pathogenicity.Methods Overexpression of the z5150 gene was used to test its toxicity.The kanamycin resistant gene flanked by homologues of target genes was amplified by PCR before being transferred into EHEC containing pKD 46 plasmid,and the target fragments were recombined into genome by Arabia sugar.The kanamycin resistance gene was removed by pFLP 2 plasmid mediated FLP site-specific recombination.Cell adhesion ability and virulence of the mutant were detected by infecting HT 29 cells and BALB/c female mice,respectively.Results and Conclusion The mutant strain EHEC △z5150 was successfully constructed.The absence of z5150 did not affect the normal growth of bacteria,but after excessive expression,it inhibited the growth.Deletion of z5150 showed significant difference in intestinal epithelial cell adhesion and pathogenicity compared with wild strain.This study provides a gene knockout technique for the study of EHEC gene function and of the relationships between RelEz5150 and EHEC pathogenicity.
ABSTRACT
Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.
La citocromo c oxidasa (COX) emplea electrones obtenidos del citocromo c para producir la reducción del oxígeno a agua. Se sabe que los electrones originan a partir del hemo del citocromo c, y entran en la COX bovina en Trp-104. También se conoce que Tyr-105, Glu-198 y Asp-158 de la subunidad II de COX, desempeñan papeles en la catálisis de la enzima, pero no hay todavía claridad en cuanto a cómo estos papeles se hallan vinculados con la transferencia de electrones. Recientemente, sugerimos que los electrones viajan del borde del hemo del citocromo c al CuA, el primer centro metálico de reacción redox de la COX, por un relé iónico hidrógeno-hidruro, usando seis residuos. Ahora, usando un enfoque similar computarizado, investigamos hasta que punto este mecanismo de iones hidrógeno/hidruro es común entre las oxidasas. Se bajaron y analizaron los dominios de unión de las estructuras cristalinas de la COX de P denitrificans, R sphaeroides, y T thermophilus, y de la quinol oxidasa de la E coli. Como en el caso de la bovina, las cuatro oxidasas tenían sólo nueve residuos de aminoácido en esa región, y tanto las secuencias como las estructuras tridimensionales presentaban un alto grado de conservación. Proponemos que estos residuos funcionan como un relé iónico hidrógeno-hidruro, participando directamente en una transferencia de electrones al CuA. Asimismo, sugerimos que este mecanismo de transferencia de electrones podría ser un rasgo común de las oxidasas.
Subject(s)
Animals , Cattle , Electron Transport Complex IV/metabolism , Cytochromes c/metabolism , Heme/chemistry , Hydrogen/metabolism , Oxidation-Reduction , Paracoccus denitrificans/enzymology , Protons , Rhodobacter sphaeroides/enzymology , Amino Acid Sequence , Thermus thermophilus/enzymology , Escherichia coli/enzymologyABSTRACT
0.05). However,compared with others segments, significantly high per centage of TA-SLN was absorbed in colon(P0.05). With the decrease of Na+ or existence of deoxycholate, T ween 80,SLS or DNP in the perfusion solution, the absorption rate increased rem arkably(P